Masses of many macromolecules of biological interest, e.g., protein molecules and others with masses of the order of 10.sup.4 to 10.sup.8 amu, are not known to high accuracy. At the same time, a need exists to analyze mixtures of macromolecules in this general mass range.
Whereas it is possible to envisage applying mass spectrometric methods such as are used in the mass range below 10.sup.4 amu for both mass determination and analysis of mixtures of molecules and atoms, a basic problem in extending these mass spectrometric techniques to macromolecular ions lies in the detection of the ions following their analysis according to their charge-to-mass ratio.
In a conventional mass spectrometer using light ions (less than 10.sup.4 amu and more normally less than 10.sup.3 amu), the ions can be detected through secondary electron emission at a surface followed by electron multiplication in conventional electron multiplier tubes. In this method of detection, the ion is accelerated through a potential drop of about 300 volts which gives the ion a velocity of the order of 10.sup.7 cm/sec. This velocity is sufficient to cause secondary electron emission when the ion strikes the surface.
If the ion is heavy, however, for example 10.sup.6 amu as opposed to 10.sup.3 amu, accelerating the ion through the same potential drop provides it with a velocity much less than 10.sup.7 cm/sec. At this lower velocity the ion is not capable of ejecting secondary electrons from the surface with a satisfactory probability. To increase the efficiency of ejection of secondary electrons, it is necessary to increase the potential through which the heavy ion is accelerated toward the surface. However, practical limitations intrude; in order to give a heavy ion sufficient velocity to cause efficient secondary electron emission at the surface the accelerating potential difference must be of the order of millions of volts and this becomes impracticable. A need thus exists for an alternative detector for heavy ions.
Our co-pending patent application Ser. No. 319,422 discloses a method for the detection of small particulate matter and macromolecules of 10.sup.3 amu or greater, wherein the particulate or macromolecule strikes a heated surface. Upon striking the surface, the particulate or macromolecule decomposes and surrenders to the surface the decomposition products which include light atoms, molecules and radicals. If these decomposition products have ionization potentials or electron affinities comparable to the work function of the surface they become surface ionized and bursts of light ions of these light decomposition products are emitted from the surface. These light ions can be detected by well known conventional means for the detection of light ions.
As pointed out in patent application Ser. No. 319,442, the ions of the decomposition products are emitted from the surface in times of the order of microseconds. If a particulate or macromolecule contains a number of decomposition products which are surface ionizable, the light ions are all emitted in times of this order, and thus a burst of ions from the surface signals the arrival of a large particulate or macromolecule at the surface. The large quantity of charge contained in this burst of ions permit its being distinguished from ions from surface ionizable impurities in the material of the hot surface through the use of conventional pulse discrimination techniques.
Experiments have been performed which indicate that the method disclosed in patent application Ser. No. 319,442 readily detects particulates in the size range below 0.1 microns (10.sup..sup.-5 cm) in diameter, through use of calibrated filters. The signal-to-noise ratio in these experiments indicate that particulates can be detected at diameters an order of magnitude less, i.e., 0.01 microns (10.sup..sup.-6 cm).
A particulate or macromolecule of this size, if it has a density of approximately 1 gm/cm.sup.3, as do biological molecules, it has a mass of approximately 10.sup..sup.-18 grams, which is equal to approximately 6 .times. 10.sup.5 amu. This is the mass range of interest for mass spectrometry of macromolcules. Thus, the methods and apparatus described in patent application Ser. No. 318,442 are applicable to detection of macromolecular ions following mass analysis.
Additional background to the invention relates to means for accomplishing the separation of ions according to their charge-to-mass ratio, prior to detection. In particular, among other means, the quadrupole mass filter invented by W. Paul and disclosed in U.S. Pat. No. 2,939,952 will accomplish the ion separation. From the well substantiated theory of quadrupole mass filters given by Professor Paul, it is readily calculable that a mass filter of pole diameter 3.6 mm, of length 30 cm, driven by an ac volage at approximately 180 kHz at a maximum amplitude of 5000 volts will analyze ions up to a mass-to-charge ratio of 10.sup.6 amu/electronic charge. The details of construction of such mass filters and the requisite voltage supplies are well within the state of the art.